Multimodal chromatography combining steric exclusion and cation exchange as an intermediate downstream step to purify yellow fever virus-like particles

Yellow fever (YF) is an hemorrhagic viral disease transmitted by infected mosquitoes, which is endemic in many African and Central/South American countries. The severe symptoms and the high mortality rate of the disease can have devastating effects in case an outbreak occurs in an area where the population is non-vaccinated. Before the current YF vaccine became available, outbreaks in cities like Barcelona (Spain) and Philadelphia (USA) led to the death of approximately 10% of the population. Recent outbreaks have shown that YF continues to be a major public health threat due to production capability issues and shortage of vaccine stockpiles, which even led to the use of an emergency fractional (1/5) dose in Africa in 2016 and in Brazil in 2018. Yellow fever virus-like particles (VLPs) represent an interesting alternative to develop a new YF vaccine. With the aim of developing an efficient and affordable process to purifiy yellow fever VLPs, in this work we developed a multimodal strategy combining cation exchange (CEX) and steric exclusion chromatography (SXC) under conditions where the product of interest does not bind to the CEX adsorber, whereas many contaminants do. In this way, the product of interest is retained just due to steric exclusion by the polyethylene glycol (PEG) added to the mobile phase. Product desorption can be achieved by decreasing PEG concentration, while contaminants remain bound to the adsorber and are eluted in the regeneration step. To the best of our knowledge, the application of such a multimodal strategy has not been published before. Please click Download on the upper right corner to see the full abstract

Hydrocyclones for single-use perfusion application

Hydrocyclones (HC) are very compact devices that promote solid-liquid separation under the action of a centrifugal field. Despite the small size, HCs have a large processing capacity and do not suffer from clogging. Therefore, several publications explored HCs as a potential cell retention device in perfusion applications in the last 20 years, but limited to non-disposable lab-scale bioreactors and to relatively low cell densities (up to ~10 million cells/mL). Even though the absence of moving parts may streamline the HC manufacturing, the performance of solid-liquid separation is highly dependent on the HC internal geometry. Said that, hydrocyclones can be produced by 3D printing, making them a promising alternative for the integration of cell retention devices in single-use bioreactor bags. The performance of hydrocyclones also depends on the attachment configuration to the bioreactor and cell concentration of the feed suspension. In this work, at first rapid batch tests were carried out to evaluate the impact of: (i) cell concentration; (ii) diameter of connector installed in the recirculation loop; and (iii) controlled harvest flow rate enabled by a peristaltic pump (520U model, Watson Marlow). The main response considered was their effect on HC separation efficiency. The stainless-steel HC2015 designed for mammalian cell separation (Pinto et al., 2008) was selected for the preliminary batch tests, and also used as a benchmark for plastic prototypes produced by 3D-printing techniques. Afterwards, the same HC2015 was installed in a 50-L single-use bag (XDR50 Xcellerex, GE Healthcare) specially customized for a perfusion cultivation with a mAb producer CHO cell line. The stainless-steel HC2015 when operating at 2.3 bar provided a total separation efficiency (Et) up to 96%, and a centrifugal separation efficiency (E´) of 82% for a CHO cell suspension at 24E6 viable cells per mL Concentrated cells recovered by the underflow port did not show decrease in viability compared to the feed suspension. The reduction of a TC connector size from 19.7 to 12.7 mm resulted in the total filling of the recirculation loop with liquid, disrupting the formation of the desirable umbrella-pattern discharge of the underflow and reducing cell retention. The use of a peristaltic pump to control the overflow flow rate equivalent to perfusion rates of 1 and 2 RV (reactor volume) per day in 40-L bioreactor working volume resulted in a reduction of the E´ values and a consequent increase of cell concentration in the harvest stream. The reduction in the separation efficiency was probably due to a disturbance of the liquid flow pattern inside the HC, since it was observed that the typical gas core coming out from the overflow was absent. These features were taken into account in the HC operation in the single-use 50-L perfusion bioreactor, and a cell concentration of 50E6 cells per mL was successfully achieved with a cell-specific perfusion rate (CSPR) as low as 20 pL per cell per day. The harvest stream consisted of a natural cell bleeding leaving the overflow outlet. Moreover, the lower cell viability and average diameter in the overflow evidenced the preferential retention of viable cells returning into the bioreactor, thus providing a healthier culture environment. A 3D-printed hydrocyclone with equivalent geometry to the stainless-steel HC2015 was made and presented slightly lower separation efficiencies. Further studies proposing materials with a smoother surface and investigating further 3D-printing techniques are currently ongoing. Pinto, R. C.V., Medronho, R. A., Castilho, L. R. (2008). Separation of CHO cells using hydrocyclones. Cytotechnology, 56(1), 57–67. doi:10.1007/s10616-007-9108-

Analyzing the fluidization of a mixture of gas-sand-biomass using cfd techniques

Fluidization taking into account the presence of the material to be gasified is a differential addressed in this study. Accordingly the solid phase was composed of a binary mixture of sand and biomass. This paper deals with the numerical simulation of a gasifier bubbling fluidized bed using CFD for the system composed of gas - biomass – sand. In order to determine the best fluidization conditions, a factorial design 23 was carried out varying the biomass particle density and diameter and the biomass percentage in the solid phase. To perform the simulations, ANSYS CFX 15.0 was used, adopting an Eulerian approach coupled to the Kinetic Theory of Granular Flow. The k- ε turbulence model was adopted. Seventeen simulations were performed setting the gas superficial velocity to 0.38 m s-1. Based on the results of the factorial design, it was possible to qualitatively identify the tests to which the system reached a bubbling fluidization (1, 2, 5, 6, 9, 11, 12 and 14). The variable with the highest significance in the model equation was the diameter of the biomass particle. Volumetric fraction profiles of gas, sand and biomass were obtained to the 17 factorial design conditions as well as a model that predicts the bed expansion. The assay that reached greater bed height (0.50 m), staying on bubbling regime, was the one with 15% biomass particles with 375 m diameter and 85% sand, indicating those are good conditions for fluidization. Please click Additional Files below to see the full abstract

Acacia wood fractionation using deep eutectic solvents: extraction, recovery, and characterization of the different fractions

The selective extraction and recovery of different lignocellulosic molecules of interest from forestry residues is increasing every day not only to satisfy the needs of driving a society toward more sustainable approaches and materials (rethinking waste as a valuable resource) but also because lignocellulosic molecules have several applications. For this purpose, the development of new sustainable and ecologically benign extraction approaches has grown significantly. Deep eutectic solvents (DESs) appear as a promising alternative for the processing and manipulation of biomass. In the present study, a DES formed using choline chloride and levulinic acid (ChCl:LA) was studied to fractionate lignocellulosic residues of acacia wood (Acacia dealbata Link), an invasive species in Portugal. Different parameters, such as temperature and extraction time, were optimized to enhance the yield and purity of recovered cellulose and lignin fractions. DESs containing LA were found to be promising solvent systems, as the hydrogen bond donor was considered relevant in relation to lignin extraction and cellulose concentration. On the other hand, the increase in temperature and extraction time increases the amount of extracted material from biomass but affects the purity of lignin. The most promising DES system, ChCELA in a ratio of 1:3, was found to not significantly depolymerize the extracted lignin, which presented a similar molecular weight to a la-aft lignin. Additionally, the P-31 NMR results revealed that the extracted lignin has a high content of phenolic OH groups, which favor its reactivity. A mixture of ChCl:LA may be considered a fully renewable solvent, and the formed DES presents good potential to fractionate wood residues.info:eu-repo/semantics/publishedVersio

Smear plus Detect-TB for a sensitive diagnosis of pulmonary tuberculosis: a cost-effectiveness analysis in an incarcerated population

Background: Prison conditions can favor the spread of tuberculosis (TB). This study aimed to evaluate in a Brazilian prison: the performance and accuracy of smear, culture and Detect-TB; performance of smear plus culture and smear plus Detect-TB, according to different TB prevalence rates; and the cost-effectiveness of these procedures for pulmonary tuberculosis (PTB) diagnosis. Methods: This paper describes a cost-effectiveness study. A decision analytic model was developed to estimate the costs and cost-effectiveness of five routine diagnostic procedures for diagnosis of PTB using sputum specimens: a) Smear alone, b) Culture alone, c) Detect-TB alone, d) Smear plus culture and e) Smear plus Detect-TB. The cost-effectiveness ratio of costs were evaluated per correctly diagnosed TB case and all procedures costs were attributed based on the procedure costs adopted by the Brazilian Public Health System. Results: A total of 294 spontaneous sputum specimens from patients suspected of having TB were analyzed. The sensibility and specificity were calculated to be 47% and 100% for smear; 93% and 100%, for culture; 74% and 95%, for Detect-TB; 96% and 100%, for smear plus culture; and 86% and 95%, for smear plus Detect-TB. The negative and positive predictive values for smear plus Detect-TB, according to different TB prevalence rates, ranged from 83 to 99% and 48 to 96%, respectively. In a cost-effectiveness analysis, smear was both less costly and less effective than the other strategies. Culture and smear plus culture were more effective but more costly than the other strategies. Smear plus Detect-TB was the most cost-effective method. Conclusions: The Detect-TB evinced to be sensitive and effective for the PTB diagnosis when applied with smear microscopy. Diagnostic methods should be improved to increase TB case detection. To support rational decisions about the implementation of such techniques, cost-effectiveness studies are essential, including in prisons, which are known for health care assessment problems

Experimental study of water droplet break up in water in oil dispersions using an apparatus that produces localized pressure drops

Oil production facilities have choke/control valves to control production and protect downstream equipment against over pressurization. This process is responsible for droplets break up and the formation of emulsions which are difficult to treat. An experimental study of water in oil dispersion droplets break up in localized pressure drop is presented. To accomplish that, an apparatus simulating a gate valve was constructed. Droplet Size Distribution (DSD) was measured by laser light scattering. Oil physical properties were controlled and three different break up models were compared with the experimental results. All experimental maximum diameters (dmax) were above Kolmogorov length scale. The results show that dmax decreases with increase of energy dissipation rate (ε) according to the relation dmax ∝ ε−0.42. The Hinze (1955, AIChE J.1, 3, 289–295) model failed to predict the experimental results, although, it was able to adjust reasonably well those points when the original proportional constant was changed. It was observed that increasing the dispersed phase concentration increases dmax due to turbulence suppression and/or coalescence phenomenon. Turbulent viscous break up model gave fairly good prediction

Experimental study of water droplet break up in water in oil dispersions using an apparatus that produces localized pressure drops

Oil production facilities have choke/control valves to control production and protect downstream equipment against over pressurization. This process is responsible for droplets break up and the formation of emulsions which are difficult to treat. An experimental study of water in oil dispersion droplets break up in localized pressure drop is presented. To accomplish that, an apparatus simulating a gate valve was constructed. Droplet Size Distribution (DSD) was measured by laser light scattering. Oil physical properties were controlled and three different break up models were compared with the experimental results. All experimental maximum diameters (dmax) were above Kolmogorov length scale. The results show that dmax decreases with increase of energy dissipation rate (ε) according to the relation dmax ∝ ε−0.42. The Hinze (1955, AIChE J.1, 3, 289–295) model failed to predict the experimental results, although, it was able to adjust reasonably well those points when the original proportional constant was changed. It was observed that increasing the dispersed phase concentration increases dmax due to turbulence suppression and/or coalescence phenomenon. Turbulent viscous break up model gave fairly good prediction

Effects of EOR chemicals and superficial gas velocity on bubble size and gas holdup of a bubble column

International audienceChemical Enhanced Oil Recovery (EOR) can boost oil extraction in offshore operations, however one of the main concerns regarding its application is how the efficiency of flotation units for treating produced water is affected. The present work thus focuses on investigating the impact of EOR chemicals on the physical properties of EOR effluents and how this can affect flotation performance parameters such as bubble size and gas holdup. Design of experiments has been used to assess the influence of polymer, surfactant and sodium chloride concentrations on bubble size and gas holdup of a laboratorial bubble column. The influence of superficial gas velocity has also been assessed together with chemicals concentrations, yet at low levels in order to avoid clusters, swarms and foam. The characterization of the synthetic effluent containing polymer, surfactant and sodium chloride has indicated that the fluid behaves as a non-Newtonian fluid, what makes separation processes in flotation cells challenging. Results showed that polymer concentration of 2000 mg/L can lead to significant increases in fluid viscosity, promote a growth of more than 40% in bubble size and only increases gas holdup when surfactant is present at high concentration. Therefore, polymers are expected to be detrimental to produced water treatment. Surfactants decrease both fluid surface tension and bubble size, increasing gas holdup. For the range studied, superficial gas velocity favors gas holdup and sodium chloride concentration seems to weakly influence bubble size and gas holdup. This work highlights the fact that changes in physical properties of produced water do modify bubble size distribution and gas holdup and this must therefore be taken into account when flotation-like systems are designed to deal with EOR effluents